爲了明确PCR産物直接測序與克隆後(如請hòu)測序結果的差異，驗證PCR産明懂物直接測序的“準确性”，將(jiāng)從兩(liǎng)個規模化豬場采集的麗鄉組織樣(yàng)品（A、B）PRRSV ORF5 PCR陽性擴增愛雜産物膠回收後(hòu)連接至pMD答火19-T載體，分别取15個陽性算鄉亞克隆單菌落，同原始PCR産物進(jìn)行測序，對(duì)兩(l個商iǎng)個樣(yàng)品各獲得的16條序列進(jìn)行比對(理從duì)分析。結果顯示：A、B兩(liǎng)組樣(yàng)品16條序列間靜視的核苷酸同源性分别爲84.2%~100%、87.4%~1書高00%，PCR産物與其15個亞克隆測序獲得序列的核苷酸同源性河行分别爲87.7%~97.3%、87.9%~木外100%。遺傳演化分析方面(miàn)：樣(裡子yàng)品A序列與其亞克隆序列被(bèi)劃分在店老譜系1、5、8，占比分别爲6.25%、87.50%、6.25%，與其12個拿制亞克隆序列同屬于譜系5；樣(yàng)品B與其亞冷快克隆序列被(bèi)劃分在譜系5、8，占比分别爲68也件.75%、31.25%，與其1那議0個亞克隆序列同屬于譜系5。結果表明，同一樣(yàng)品中PRRSV ORF森多5序列具有多樣(yàng)性，不同樣(yàng)品的P小電RRSV ORF5序列豐富度不同。結果提示，在生産實際少習中，通過(guò)PCR對(duì)PRRSV O業微RF5産物進(jìn)行直接測序，對(duì)于了解場内流行毒株具有一定的放日指示意義。
Comparison of Sequencing Results河笑 between PRRSV ORF5 Amp村服lification Products and Its Cloning
In order to clar地理ify the differen喝雜ce between direct s醫離equencing and post c志通loning sequencing頻分 of PCR products，and ver要船ify the“accuracy”of the for生呢mer，PRRSV ORF5 PCR posi購拍tive amplified product glue of the小紙 tissue samples（A，B）collec窗讀ted from two inten線小sive swine farms were recycl國看ed and then connected to pMD19分國-T vector，15 positive 資土subclone single colonies were資弟 respectively taken and sequ她報enced with the original PCR products，時志then 16 sequences obtaine器請d from each sample were 花用compared and anal體綠yzed. The resul喝相ts showed that the nucleotide hom雨多ology between th理技e 16 sequences in t和書he two groups were 謝是from 84.2% to 100% 高好and from 87.4% to 100%，respec習到tively，and that 理麗of PCR products and their 15 subcl湖公one sequences were from 87.7% to 9也志7.3% and from 87.9% to 100%秒秒，respectively. For g微通enetic evolution兒窗 analysis，the se你黑quence of sample A and會開 its subclone sequences were div文問ided into lineage些從s 1，5 and 8，accounting f媽章or 6.25%，87.50% and 6.25%，respe兵信ctively，sample A together with 吃身its 12 subclone 她請sequences belonged to lineages技習 5；sample B and its subclone sequen廠村ces were divided議兵 into lineages 5 and 8，accounting f嗎國or 68.75% and 31.25關低%，respectively，sample B together with 又你its 10 subclone sequences be會廠longed to lineages也北 5. In conclusion，PRRSV OR麗討F5 sequence were 錢道diverse in the same sample b吧訊ut with different abun件子dance in different sampl弟討es. Therefore，direct se自照quencing of PRRSV ORF5 products by PCR綠坐 contributed to identi男小fying the prevalent st黃對rains within a farm.
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